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Notes: Sunlight is essential for human life but we need to avoid overexposure to the sun. Chronic exposure of the skin to UV radiation leads to photoageing (sunburn, wrinkles, spots, freckles, skin texture changes, and dilated blood vessels), immunosuppression, and sometime more serious lesions. UV radiation also causes DNA damage, which is a critical event in skin photoageing and photocarcinogenesis [1]. Exposure to UV light induces wide range of DNA lesions on skin cells by direct absorption (UVB), or via oxidative stress (UVA and UVB). We choose to work on healthy Chinese women skin because it is demonstrated that Asian people developed less skin cancer than Caucasian, and few studies have been done on DNA damage in the skin of Asian subjects [2, 3].The formation of 8-hydroxy-2'deoxyguanosine (8-oxo-dG) is a major type of DNA lesion resulting from oxidative stress. It is a biological marker of oxidative stress on DNA. It causes the transversion of G : C to T : A during DNA replication, which occurs frequently in some genes in human skin lesions on areas exposed to sunlight [4]. Skin cells have evolved several DNA repair mechanisms to counteract immediately the deleterious effects of such lesions that can lead to genomic instability. These repair pathways are present in all living cells and are extremely well conserved. P53 is a phosphoprotein that is activated by stress, up-regulates DNA repair enzymes, participates directly in DNA repair, blocks cell cycles during DNA repair, and induces apoptosis in critically damaged cells. After exposure to UV, basal keratinocytes repair damaged DNA whereas differentiating keratinocytes undergo cell death, both processes are regulated by p53 [5].Repeated exposure of the skin to UV light induces pigmentation and thickening of the epidermis, both of which help to increase tolerance of sunlight. Some studies have shown that this photo-adaptation can help preserve epidermal DNA from UV injury [2, 6–8]. However, as most of these studies were performed by exposing the skin to a single or several acute bursts of radiation, they do not necessarily reflect the effect of chronic exposure to sunlight. The first aim of this study on 15 healthy Chinese women was to determine the effects of chronic exposure to sunlight on DNA damage in the skin. We compared, on sun-protected and sun-exposed skin of the same patient, the amounts of p53 protein and 8-oxo-dG determined by immunohistochemistry method. Blood samples from these women were used to measure their anti-oxidant stress status and hence their intrinsic defence capacities.We also evaluated how changes in the skin induced by chronic exposure to sunlight helped preserve DNA from an acute radiation. The two areas (chronically exposed to and protected from sunlight) were exposed to a relatively low dose of 1 minimal erythema dose (MED). The amounts of the markers (p53 protein and 8-oxo-dG) and the responses of the two areas were compared 24 h after exposure.〈section xml:id="abs1-2"〉〈title type="main"〉Methods〈section xml:id="abs1-3"〉〈title type="main"〉VolunteersVolunteers were recruited and biopsies removed at the Laboratoires Dermexpert (Paris, France), in accordance with international ethical procedures.Volunteers completed a questionnaire that allowed us to estimate their skin sensitivity, phototype, life styles and eating habits. The skin of patients was clinically evaluated by a dermatologist. Blood and urine were taken and analysed. Skin punch biopsies (3 mm) were taken from the anterior surface of the upper arm (protected area) and the posterior surface of the forearm (exposed area). MED was determined. Two zones (one exposed and one protected) on the other arm were irradiated at 1 MED. Punch biopsies (3 mm) were taken from the two irradiated zones 24 h after irradiation.〈section xml:id="abs1-4"〉〈title type="main"〉MED determinationThe MED values were determined using a multiport solar light (601 model). The anterior forearm test site was exposed to six increasing doses (13.44; 16.8; 21.0; 26.46; 32.76 and 40.95 mJ cm–²). The skin reaction was evaluated visually 24 h later. The lowest dose of UV energy that caused a perceptible demarcated erythema was considered to be 1 MED. Those subjects that had a very mild erythema after the maximum dose (40.95 mJ cm–²) were to be irradiated at 51.24 mJ cm–².〈section xml:id="abs1-5"〉〈title type="main"〉Oxidative stress statusSamples of blood and morning urine were carried out from fasting subjects. The blood samples were used to measure the following parameters: antioxidants (vitamin C, vitamin E, carotenoids, glutathione, thiol proteins, uric acid, total antioxidant capacity), trace metals (selenium, copper, zinc), markers of oxidative stress (lipid peroxidation), and iron status (free iron, ferritin, transferrin). The urine samples were used to assay 8-oxo-dG. Assays were performed by Probiox SA (Liege, Belgium) [9, 10].〈section xml:id="abs1-6"〉〈title type="main"〉ImmunohistochemistryThe punch biopsies were immediately placed in 10% neutral buffered formalin (4% formaldehyde) and fixed for 24 h at ambient temperature. They were then embedded in fresh wax and p53 and 8-oxo-dG detected immunohistochemically using BP53-12-1 and N45.1 monoclonal antibodies respectively [11]. Two hundred epidermal cells were counted per sample and the numbers of p53 or 8-oxo-dG positive and negative cells were determined.The statistical methods used were analysis of variance (anova) and linear regression, and all data were assessed for statistical significance (P 〈 0.05).〈section xml:id="abs1-7"〉〈title type="main"〉ResultsThe 15 healthy Chinese women living in France with a age range of 31--43 years (mean: 36 years). The clinical evaluation of the skin performed by a dermatologist and based on wrinkles, heliodermy and pigmentation disorders, showed that all 15 women had similar degree of cutaneous photoageing. Similarly, analysis of global anti-oxidant stress status showed that all 15 women had the same antioxidant potential, providing same intrinsic defence capacities.The amounts of 8-oxo-dG in protected (six to 64 positive cells per 200 epidermal cells; mean: 18.8) and exposed areas (two to 40 positive cells per 200 epidermal cells; mean: 17.1) varied greatly from one individual to another. No difference in the 8-oxo-dG contents was observed between the two areas (〈link href="#f1-16"〉Fig. 1). And there was no statistical correlation between the 8-oxo-dG in the skin and in the urine. The large differences in 8-oxo-dG in the skin between individuals were not correlated with any of the life style parameters in the questionnaire or with any of the blood antioxidant parameters.〈figure xml:id="f1-16"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_16_16:ICS_254_f1-16"/〉Epidermal detection of p53 protein and 8-oxo-dG in sun-protected and chronically sun-exposed skin. Data are the mean of 15 biopsies. These was no statistical difference in the p53 or 8-oxo-dG in the protected and exposed areas.Immunochemical staining for p53 in biopsies of protected skin from all individuals showed one to seven (mean 1.8) stained cells per 200 cells in the epidermis. The skin chronically exposed to sunlight had the same number of p53-stained cells (mean: 1.7) for all women (〈link href="#f1-16"〉Fig. 1). P53-positive nuclei were found in the basal and suprabasal cells of the epidermis. Thus, the sun-protected and sun-exposed areas of skin contained the same amounts of both 8-oxodG and p53.The second part of the study compared the responses of protected and chronically exposed samples of skin from the same patient to a single acute UV radiation. The skin areas of all 15 subjects were exposed to 1 MED and the removal of DNA lesions (8-oxo-dG) and p53 were quantified 24 h later. The MED were similar for 15 volunteers (40.95 or 51.24 mJ cm–²), suggesting that they were all similarly sensitive to sunlight. There was erythema in both areas 24 h after radiation, but it was more

Notes: The extent of changes with aging depends largely on how much the skin is exposed to sunlight and also on the genetic disposition of the individual. There are thus two main processes, extrinsic aging because of environmental stress, and intrinsic genetically programmed aging [1]. Hence, the processes of aging depends on a person's ethnic origin and the part of the world in which they live.〈section xml:id="abs1-1"〉〈title type="main"〉MethodsThe present study investigates the influence of age and exposure to sunlight on the facial skin of 31 healthy Japanese women, aged 20–78 years old, living in Osaka city. Skin samples were obtained during plastic surgery from the face, from areas exposed to varying intensities of sunlight (forehead, cheek, nose, upper eyelid). Samples were fixed in formalin and embedded in paraffin. Sections (5–7 μm) were cut and stained with hematoxylin–phloxyn–safran, orcein or Masson's trichrome. Others were immunostained for p63, β1-integrin, type IV collagen CD1a and AQP3. Statistical analysis of quantitative and qualitative parameters were performed by analysis of variance (anova) with linear regressions, and the chi-squared test.〈section xml:id="abs1-2"〉〈title type="main"〉Results and discussion〈section xml:id="abs1-3"〉〈title type="main"〉Epidermis and dermal-epidermal junction: histological organizationWe confirmed that the whole living epidermis becomes thinner with increasing age, with an average decrease of about 5 μm per decade. This thinning is mainly because of a significant reduction in the number of keratinocyte layers. The thinning of the epidermis and the reduction of the keratinocytes layers in Japanese skin do not seem to be reflected in the thickness of the stratum corneum, which appears to remain constant whatever the age in the sample studied.The epidermal papillary structures also became flatter with age, associated with an increase in the thickness of the dermal–epidermal junction (DEJ). Thus, in addition to the loss of epidermal cells, the epidermis and dermis become less overlapped, so reducing the surface area for exchange between the two compartments. The DEJ also become thicker as Japanese ages and the expression of type IV collagen, the main constituent of the lamina densa and anchoring plaques is reduced in the most photoexposed skin areas. This accounts for the major changes in the function and molecular structure of DEJ components, as in aged Caucasian skin [2].〈section xml:id="abs1-4"〉〈title type="main"〉Keratinocyte growth and differentiation –β1-integrin and p63There is no doubt that the rate of cell turnover decreases in the flat aged epidermis, as indicated by the smaller number of proliferative cells [3, 4]. This study focused on two key regulatory proteins. One was p63, that is involved in maintaining the proliferative potential of basal keratinocytes and blocking their calcium-induced differentiation [5]. The other was β1-integrin, an adhesion protein present in basal keratinocytes and linked to their clonogenic potential [6].We found p63-positive cells in the basal layer of the epidermis and in the suprabasal layers (〈link href="#f1-7"〉Fig. 1a), in agreement with others [7]. The used pan anti-p63 antibody suggest that other isoforms of the protein in addition to the major ΔNp63α mainly expressed in the basal cells, could take part to other functions like differentiation in the suprabasal layers [5]. The great interindividual variation in the staining intensity for p63 in the samples studied made it impossible to detect significant changes in the number of p63-positive cells with age. Only an increase of p63 was observed in photoexposed areas compared to others within this case, a possible relation to the epidermal thickening.〈figure xml:id="f1-7"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_7_7:ICS_254_f1-7"/〉Immunostaining of Japanese facial skin showing AQP3 expression at the plasma membrane of keratinocytes throughout the living epidermis (a) and the age-related change in AQP3 (b).β1-integrin was detected only in the basal keratinocytes, and the staining intensity varied from one segment of the basal layer to another. We evaluated the length of immune-labeled epidermis and the intensity of labeling in each fragment, using an arbitrary colorimetric scale (0–5). We observed a significant decrease in the intermediate intensities (equal to 2 only) with age in the zone least exposed to sunlight. This is consistent with the loss of adhesive properties of freshly isolated epidermal cells in aged skin [2] and the existence of different pools of basal keratinocytes [6]. It suggests that the effect of aging could affect particularly on the transit amplifying cells containing intermediate quantities of β1-integrin.〈section xml:id="abs1-5"〉〈title type="main"〉Osmotic and water homeostasis – aquaporin-3In addition to the stratum corneum (SC) that limits transepidermal water loss, the osmotic equilibrium inside the epidermis and hydration is controlled by the aquaglyceroporins 3 (AQP3) [8, 9]. Immunostaining for AQP-3 confirmed the presence of the protein in the plasma membranes of keratinocytes throughout the epidermis, together with AQP-3 cytoplasmic expression of the basal layer cells (〈link href="#f1-7"〉Fig. 1a). There was no immunostaining in the SC (〈link href="#f1-7"〉Fig. 1a) to retain water in the epidermis via the tight junction proteins [10], so maintaining the water-lipid barrier within the SC. The immunostaining for AQP3 decreased significantly with the skin age (〈link href="#f1-7"〉Fig. 1b), but there was no significant difference between areas exposed to sunlight and those not exposed. This suggests that there is an overall reduction in the natural hydration potential as Japanese facial epidermis ages.〈section xml:id="abs1-6"〉〈title type="main"〉The immune system – epidermal CD1a-positive cellsEpidermal dendritic cells, mainly Langerhans cells, control the skin immune system. These cells are CD1a positive (CD1a+). Immunostaining showed a major population of highly dendritic cells throughout the epidermis of all the Japanese skin samples. It also showed that the areas most exposed to sunlight (cheeks, forehead and nose) had significantly more CD1a+ cells than less exposed areas (upper eyelid) (〈link href="#f2-7"〉Fig. 2a). This confirms previous findings on the wrinkling of area of Caucasian facial skin after chronic exposure to UV light [11]. The number of CD1a+ cells in less exposed areas of skin increased significantly with donor age (〈link href="#f2-7"〉Fig. 2b). This shows that chronological aging and exposure to sunlight give rise to an epidermis which cellular immune homeostasis is perturbed.〈figure xml:id="f2-7"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_7_7:ICS_254_f2-7"/〉Immunostaining for CD1a in Japanese facial skin: numbers of CD1a-positive (CD1a+) epidermal cells per mm of dermal–epidermal junction (DEJ) in skin exposed to sunlight and skin from protected areas (a), and sun-protected area of facial skin from subjects of different ages (b).〈section xml:id="abs1-7"〉〈title type="main"〉The pigmentation system – melanocytes and melanin depositsPigmentation is part of the protective system developed by the skin epidermis, but unwanted pigmentation is one of the first signs of aging and has a major social impact. The pigmentation of the facial skin of Japanese women increases with age, and spots develop, particularly on the cheeks [12]. We found a significantly greater density of melanocytes in the epidermis of older donors in both sun-exposed and protected areas. But there were significantly fewer melanocytes in the areas exposed to sunlight than in protected areas. As expected, there were greater deposits of melanin in keratinocytes in the regions exposed to sunlight. This suggests that older donors had a pool of highly active melanocytes in areas of skin exposed to sunlight, with a high rate of melanin transfer to keratinocytes and/or reduced keratinoc

Notes: The skin provides a physical barrier between the host and the environment, in particular against solar radiations. Ultra-violet (UV) radiation exposure is associated with an increased risk of skin aging and carcinogenesis. The UV light that reaches the surface of the earth is divided into UVA (320–400 nm) and UVB (290–320 nm) components that have distinct biological effects. UVA is the predominant component of solar UV radiation. Although it has been considered to be only weakly carcinogenic, it causes aging and wrinkling of the skin and penetrates more deeply into the skin to reach the dermis. Hence fibroblasts are the main cell targets for UVA radiation. UVA affects cellular DNA integrity by the activation of photosensitizers that consequently generate reactive oxygen species (ROS). Among them superoxide anion (O2°–), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl radical (OH°) can react with DNA bases or sugar and induce oxidative DNA damage and strand breaks.The comet assay (known also as the single cell gel electrophoresis assay) is a popular method that allows the microscopic detection of strand breaks and oxidative damage in DNA [1]. It is based on the analysis of migration in alkaline conditions of the DNA of individual cells lysed in an agarose gel spread onto a microscope slide. Denatured DNA comes unwound at the sites of strand breaks and migrates faster than the supercoiled fraction. It gives the DNA supercoiled structure the appearance of a comet. The numbers of strand breaks (SB) and alkali-labile sites (ALS) are evaluated by the measurement of the percentage of DNA that migrates into the tail of the comet (Tail DNA). The specificity of the method can be extended to the detection of oxidated purines (mainly 8-oxodGuo) by the addition of a step in which DNA is digested by a repair enzyme (Fpg: formamido pyrimidine DNA-N-glycosylase) that specifically excises the last oxidative damage [2]. The comet assay is also a convenient method to follow the repair of damaged DNA. Non-repaired lesions and transient strand breaks may be monitored during incubation after application of a stress.We have used this very sensitive and specific method to compare the sensitivity to oxidative stress (H2O2 exposition)of human dermal fibroblasts established from sun-exposed (SE) and sun-protected (SP) female Japanese skin. We evaluated the amount of initial damage and the repair capacities (SSB, ALS, and Fpg sites) of these cells.〈section xml:id="abs1-2"〉〈title type="main"〉Materials and methods〈section xml:id="abs1-3"〉〈title type="main"〉SubjectsVolunteer recruitment and biopsy removal were performed at Laboratoire DermExpert (Paris, France) in accordance with ethical procedures. The group consisted of 10 healthy Japanese women living in France with age ranging from 30 to 45 years (mean: 36 years). Clinical evaluations and biopsies were performed by a dermatologist on each subject.〈section xml:id="abs1-4"〉〈title type="main"〉Cell cultureThe cultures of human dermal fibroblasts were established by outgrowth of 3 mm punches taken from sun-exposed (SE) and sun-protected (SP) parts of the forearm of the 10 volunteers. The cells were cryopreserved at the second passage and subsequently cultured for the experiments before passage 5.〈section xml:id="abs1-5"〉〈title type="main"〉Comet assayThe comet experiments were performed as described in [3] using sub-confluent cells. The cells were submitted to H2O2 exposition (20 mM) for 5 min at 4°C in PBS. They were then re-fed with the reserved culture medium and transferred to the CO2 incubator at 37°C to allow DNA repair. The cells were processed for the comet experiments at different times after the stress over an 8-h period.The viability of the cells was monitored by the MTT test 24 h after the H2O2 treatment. Approximately 85% of cells remained viable.We used the Komet 3.0 software from Kinetic Imaging to analyse the slides. Fifty nuclei were scored per slide and duplicate slides were processed for each experimental point. The amount of damage was evaluated by the Tail DNA (percentage of DNA in the tail of the comet). Four repair curves (Tail DNA as a function of repair time) were obtained for each donor, for SSB + ALS and for Fpg sites in both SE and SP cells.〈section xml:id="abs1-6"〉〈title type="main"〉StatisticsLinear regression curves were established for the correlation between the amount of residual damage and age. Significant differences are noted by * on the graph. anova analyses were conducted to assess the correlation between the % of residual damage and life habits.〈section xml:id="abs1-7"〉〈title type="main"〉ResultsTypical images of comets are shown in 〈link href="#f1-18"〉Fig. 1.〈figure xml:id="f1-18"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f1-18"/〉Fluorescent microscope imaging of non-damaged and damaged DNA after the electrophoresis in alkaline medium and ethidium bromide staining.For the 10 subjects, comet assay results revealed that the initial amount of damage (SSB + ALS, Fpg sites) and the damage induction by the oxidative stress were equivalent in cells from SE and SP area.Repair of SSB + ALS was significantly faster in SP cells compared with SE cells at 1 h after stress. Moreover, SP cells totally repaired the damage in 2 h while it took 4 h for the SE cells (〈link href="#f2-18"〉Fig. 2). For repair of Fpg sites, no difference was observed between SP and SE cells. It is important to stress that 8 h after the stress, return to the initial level of damage was not observed, either in the SE cells or in the SP cells.〈figure xml:id="f2-18"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f2-18"/〉Repair of SSB and ALS for SE and SP fibroblasts (mean of results obtained for each cell line). Statistically significant difference (*P 〈 0.05) between SE and SP cells.Using the clinical evaluations made by a dermatologist, we looked at the correlations that could be statistically established between the damage repair kinetics and age, skin features and life habits for each donor.Striking features appeared concerning the SP fibroblasts: the repair kinetics of the damage (SSB + ALS and Fpg sites) was strongly inversely correlated to the age of the donors. Hence, the fibroblasts from older donors had the highest levels of damage after 4 h repair (〈link href="#f3-18"〉Fig. 3a and b; r = 0.85 and 0.71 respectively). On the contrary, no correlation existed in SE cells.〈figure xml:id="f3-18"〉3〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f3-18"/〉(a) Residual (SSB + ALS) as a function of age after 4 h repair, for SP cells. (b) Residual Fpg sites as a function of age after 4 h repair, for SP cells.Repair was slower and residual damage higher for smokers or former smokers (n = 3) than for donors who had never smoked (n = 7). On the contrary, no correlation was established in SE cells.The level of overall sun exposure of the donors influenced the amount of oxidative residual damage (Fpg sites). In SE cells, residual Fpg sites were higher in cells from donors with heavy sun exposure habits. SP cells coming from donors with little sun exposure behaviour (assimilated to never exposed cells) tended to have higher Fpg-sensitive residual sites than SP fibroblasts from heavy exposed donors (assimilated to moderately exposed cells).There was no correlation between DNA repair and heliodermy, phototype, skin tanning index. Correlation with life habits questionnaire showed no influence of food consuming (rice, tea, alcohol, etc.) on DNA repair.〈section xml:id="abs1-8"〉〈title type="main"〉Discussion and conclusionAs expected, the response of the normal dermal fibroblasts, taken from sun-exposed and sun-protected skin of Japanese women, to H2O2 treatment showed inter-individual variations. Fibroblasts from SE and SP area did not display any difference in their sensitivity to the action of H2O2 stress. Although the in vitro subculture could era

Notes: Although some data on the skins of Japanese, Korean and Chinese people have appeared recently, few studies have examined the skin of south-east Asians.The population of the Philippines results for centuries of intermarriage between Chinese, Spanish and Malays. Filipino skin is different from that of Malayan Indonesians or Malaysians.Because little is known about how Filipinos skin changes with aging, we have collected data on the clinical and histological characteristics of the skin of Filipino women living in Manila. We recorded skin colour, pigmentation disorders, skin hydration, firmness, slackness, wrinkle status and carried out a histological and immunochemical evaluation of the difference between skin exposed to and protected from sunlight.〈section xml:id="abs1-1"〉〈title type="main"〉MethodsA total of 30 healthy Filipino women (mean age: 44 years) took part in this study of the clinical and histological features of their skin, and a comparison of areas protected from and exposed to sunlight was performed. All gave their informed consent. The subjects were assigned to one of the five age groups (20–30, 30–40, 40–50, 50–60 and 〉60). Overall, 52% had a dry skin and 48% had oily skin. Twelve women were in menopause. According to the Fitzpatrick classification [1], 47% of the skins were phototype IV, 50% were phototype V and 3% were phototype VI. Eleven women (37%) were former smokers and five (16%) still smoked.Facial skin colour, hydration and wrinkles were all assessed by a dermatologist. The overall severity of facial wrinkles and pigmentation brown spots were photograded using the Jin Ho Chung photograding scale [2]. Skin elasticity, firmness and slackening (face ovale shape) were also evaluated on using a scale of 0-10 [3]. The colour (Mexameter MX18) and hydration (corneometer CM820) of the skin on the left upper cheek were measured and skin replicas (Silflo/silicone resin) of the right eye contour were made. Image analysis was used to measure the number of wrinkles, total wrinkled area (mm²), total length of wrinkles (mm), mean length of wrinkles (mm) and mean depth of wrinkles (μm).Data were analysed by analysis of variance (anova) and linear regression statistical significance was taken as P 〈 0.05.Punch biopsies (4 mm) were taken from photoprotected areas (buttocks) and photoexposed regions (preauricular face). Samples were fixed in formalin and embedded in paraffin for histological and immunohistochemical evaluation. Sections were stained with Fontana Masson, Masson trichrome, orcein, haematoxylin-phloxin-safran (HPS) by standard procedures. Immunohistochemistry was carried out to detect type IV collagen and metallothionein expression.〈section xml:id="abs1-2"〉〈title type="main"〉Results〈section xml:id="abs1-3"〉〈title type="main"〉Clinical evaluationThere was a significant change in the severity of facial wrinkles with age between the women of three age groups, 20–30, 31–60 and 〉60 years. Women aged 20–30 years had very few if any wrinkles (mean grade: 0.7), those aged 31–60 years had a similar degree of wrinkling (mean grade: 2.8), while those aged over 60 years had significantly more severe wrinkles (mean grade: 4.4). The crow's feet area was the major site of wrinkles in all age groups.Women aged less than 30 years had no facial dyspigmentation (mean grade: 0.2). Those aged 31–50 years had a similar degree of pigmentation spots (mean grade: 1.25). Women over 50 years had significantly more severe dyspigmentation (mean grade: 2.2) (〈link href="#f1-6"〉Fig. 1). The patterns of facial dyspigmentation were similar in all the age groups. Women younger than 30 years had no spots on their hands; those aged 31–50 years had a similar degree of pigmentation spots (mean grade: 0.6); while those over 50 years old had significantly more severe dyspigmentation of their hands (mean grade: 1.25). The overall distribution of the spots on the face (cheeks, periocular and forehead) was similar to that found for Asian women. The hands had fewer spots than the face, as found for Chinese women.〈figure xml:id="f1-6"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_6_6:ICS_254_f1-6"/〉Photograding of facial pigmentation.The skin of the women studied showed significantly decreases in elasticity and firmness and increased slackening with age.〈section xml:id="abs1-4"〉〈title type="main"〉Skin featuresMexameter measurements showed no correlation between the erythemal index and age all the women had comparable erythemal indexes. However, the melanin index seemed to increase with age. There was a significant difference in the melanin indexes for 20–30-year-old women and women over 60 years (P ?≤ 0.05), but no difference between the indexes for women aged 20–30 and those aged 40–60. Skin hydration measured with corneometer decreased with age, being significantly different between women aged 20–40 and those 〉60. The total wrinkled area, total length of wrinkles and mean depth of wrinkles all increased significantly with age, while the number and mean length of wrinkles did not.〈section xml:id="abs1-5"〉〈title type="main"〉HistologyThe histology of the skin from areas protected from sunlight and areas exposed to sunlight was compared. Masson trichrome staining of protected skin showed a decrease in epidermis thickness with age, which was much more sever in areas exposed to sunlight. In contrast, the stratum corneum was thinner in photo-exposed areas.The structure of the keratinocytes appeared to be normal, with the overall organisation and epidermal differentiation being better and more regular in areas protected from sunlight. The basal layer was more continuous and regular and the granulous layer slightly thicker. Fontana Masson staining showed no difference in distribution of melanin in areas exposed to sunlight with aging.The collagen in dermo epidermal junction (DEJ) was more dense in the sun-exposed areas of older patients. There was a general flattening of the DEJ in the sun-protected skin of older patient. This flattening was also increased in skin exposed to sunlight. These findings are similar to those of other skin aging studies [4, 5]. Immunohistochemistry showed that the amount of type IV collagen did not vary with age or exposure to sunlight. There was also no correlation between the amount of type IV collagen and skin elasticity, firmness, slackening, or the number and length of wrinkles. However the dermis of skin exposed to sunlight had heavier deposits of elastotic material (Orcein staining). The network of elastic fibers in the superficial papillary dermis (oxytalan fibers) of skin exposed to sunlight was diminished (­50%) or absent, but it was present in skin protected from sunlight (〈link href="#f2-6"〉Fig. 2).〈figure xml:id="f2-6"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_6_6:ICS_254_f2-6"/〉Orcein staining. (A) age: 42 years, sun-protected; (B) age: 42 years, sun-exposed; (C) age: 64 years, sun-protected; (D) age: 64 years, sun-exposed. e, epidermis; d, dermis.The metallothioneins (MTs) are low molecular weight cysteine-rich, proteins that bind heavy metals; they are produced in response to a variety of stress signals [6, 7]. There are no published data on the effect of chronic exposure to UV light on metallothioneins. We therefore measured their abundance in the skin of six subjects, in areas protected from sunlight and areas exposed to sunlight (〈link href="#f3-6"〉Fig. 3). The six chosen subjects had similar good skin tolerance of sunlight. Metallothionein expression was restricted to the keratinocytes and some dermal cells. The basal layer consistently immunostained more intensely for MT than did the suprabasal layers. There was MT immunoreactivity in both the cytoplasm and nucleus of keratinocytes. The numbers of MT-positive cells in the epidermis of areas exposed to sunlight and those protected from the sun were significantly different.〈figure xml:id="f3-6"〉3〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254

Notes: The biological effects of hormones on human melanocytes are still being investigated. Several kinds of hormones have mitogenic and/or melanogenic effects on human melanocytes. For example, melanotropic hormones, such as α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotrophic hormone (ACTH), were shown to induce skin darkening some time ago, and more recently, the mechanism of up-regulation of tyrosinase activity in human normal melanocytes by melanotropins through the cyclic AMP (cAMP) signaling pathway has been reported. cAMP is now recognized to play a key role in the regulation of skin pigmentation. α-MSH is known to elevate intracellular cAMP levels through the melanocortin 1 receptor (MC1R) and this plays a critical role in the regulation of melanogenesis. Recently it has been recognized that cAMP elevating agents up-regulate transcription of the MITF gene that in turn stimulates expression of the tyrosinase gene via MITF binding to its M-box.Solar lentigo is a benign pigmented spot on sun-exposed skin especially on the back of the hands, arms and on the forehead. We have compared melanogenesis histological parameters in solar lentigo and in normal skin of Japanese women. We chose to analyze melanin distribution and expression of α-MSH as mediators of human pigmentation, in solar lentigo and normal skin biopsies of photo-exposed dorsal forearm. Punch biopsies were fixed with 4% formaldehyde and sections were stained with Fontana-Masson technique to evaluate melanin distribution. Immunohistochemistry was carried out to demonstrate α-MSH protein expression using rabbit polyclonal antibody anti-human α-MSH. Melanin distribution and relative expression of α-MSH were analyzed and quantified using semiquantitative computer-assisted image analysis. With this technique, we found that solar lentigo skin possessed 60% more α-MSH expression than normal skin (〈link href="#f1-11"〉Fig. 1).〈figure xml:id="f1-11"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_11_11:ICS_254_f1-11"/〉Quantification of alpha MSH expression in solar lentigo and normal skin for 10 japanese subjects. Immunolocalization of alpha MSH in solar lentigo and normal skin. (a) Representative image of alpha MSH expression in solar lentigo. (b) Representative image of alpha MSH expression in normal skin. Alpha MSH expression was determined from the integrated intensity given areas of epidermis reported to the total surface of epidermis in each section. Four selected areas of each section were photographed and quantified. Statistical differences were evaluated using the paired Student's t-test. Data are presented as the mean of 10 biopsies.While it has been reported that human skin color is changed by castration or by injection of androgen and a recent in vitro study in contrast suggested that testosterone might reduce tyrosinase activities in human melanocytes through the decrease of the mRNA expression of MC1R, the precise mechanism involved in the regulation of skin pigmentation by androgens has remained unclear. To date, there have been few reports about the effects of androgens on human melanocytes. In a previous study, we demonstrated that human genital melanocytes are androgen target cells. Those melanocytes had androgen receptors (ARs) in their nuclei as detected by immunohistochemistry and they also had high levels of 5α -reductase activities, the enzyme that converts testosterone to its more active form, 5α-dihydrotestosterone (DHT). R1881 (methyltrienolone, a potent synthetic androgen) stimulated tyrosinase activity when melanocytes were cultured continuously with TPA, hydrocortisone and BPE and then were treated with FBS [1]. When melanocytes were deprived of FBS and BPE for a prolonged time, the effects of androgen on tyrosinase activity were reduced. Furthermore, when cultured without FBS, BPE or TPA, prolonged exposure to R1881 down-regulated tyrosinase activity when added together with FBS and BPE. These data suggest that FBS or BPE contain elements that inhibit tyrosinase activity when contacting melanocytes even if only briefly [2].Recent advances in the understanding of androgen signaling indicate the specific membrane-signaling pathway involved in Sex hormone-binding globulin (SHBG) as well as in the androgen-AR mechanism, a classical pathway. SHBG is a glycoprotein found in human plasma that possesses a high affinity for binding of sex hormones. It has been recognized that unbound, free steroids function on target cells through their intracellular receptors. Recently, it has been speculated that SHBG participates in steroid signaling pathways by binding to SHBG-receptors (RSHBG) located on the target cell membranes. SHBG that is bound to a steroid cannot bind to its receptor, but free SHBG that first binds to the RSHBG can subsequently bind steroids. Several studies on human tissues and cultured cells, such as prostate and breast cancer cells, have revealed that the SHGB–RSHBG complex is involved in the steroid-activated signal transduction pathway that results in the accumulation of intracellular cAMP. SHBG–RSHBG complexes on the cell membrane combine with some types of sex hormones and then induce the accumulation of intracellular cAMP in those tissues and cells. Further, some steroids antagonize the activation of SHBG–RSHBG induced by other steroids. FBS contains high endogenous levels of SHBG, which can confuse the study of steroid effects. We formed the hypothesis that androgen function in melanocytes is effected through the cell membrane signaling pathway via the SHBG–RSHBG complex. Immunohistochemistry was used to demonstrate the co-staining of SHBG and testosterone with specific monoclonal antibodies. There was a dramatic increase in positive staining following SHBG pre-treatment. Generally, staining for testosterone is much stronger when staining for SHBG is stronger. These immunohistochemical results strongly suggest the existence of the RSHBG on the surface of normal human melanocytes.The responses of intracellular levels of cAMP to various androgens and hydrocortisone were studied. After the saturation of RSHBG on the cell membranes by SHBG and then washing to remove excess SHBG, a decrease in intracellular cAMP levels within 30 min of incubation with testosterone was observed in a dose-dependent manner, but not in the absence of SHBG pre-treatment. Those findings suggest the possibility that normal human melanocytes might utilize the cell membrane transduction pathway of androgen via the SHBG–RSHBG complex to regulate intracellular cAMP. They further suggest that strong androgens, such as testosterone and DHT, might act as antagonists in this pathway. We have already reported that human melanocytes possess significant levels of 5α -reductase activities and that testosterone is predominantly metabolized to DHT, a stronger androgen. DHT might work as a negative feedback factor against cAMP accumulation via the SHBG–RSHBG complex, since the affinity of DHT to SHBG is higher than that of testosterone.Tyrosinase activity was decreased by the physiological concentration of testosterone after 4 days of treatment. As the decreases in tyrosinase activity coincided with decreases of intracellular cAMP, those intracellular cAMP levels (regulated through cell membrane signaling) might regulate tyrosinase activity influenced by androgens after treatment with SHBG. Tyrosinase activity was stimulated by R1881 when the melanocytes were cultured continuously in medium containing FBS and hydrocortisone. FBS and hydrocortisone that are endogenous in the medium might work against the effect of strong androgens that may be antagonists in the cell membrane signaling pathway, because high concentrations of unbound SHBG found in FBS can easily bind to the cell surface and high concentrations of hydrocortisone added to the culture medium might have already occupied the SHBG–RSHBG complex on the cell membrane.Further, we studied the expression of tyrosinase mRNA

Notes: In humans, pigmentation of the skin results from the synthesis and the distribution of melanin pigments. Epidermal melanocytes, residing at the basal layer of epidermis, are neural crest-derived skin cells responsible for melanin pigment production [1]. Tyrosine and l-dihydroxyphenylalanine (L-Dopa) serve as precursors for this complex biopolymer and darkened skin color is the result of increased and redistributed epidermal melanin [2]. The basal level of pigmentation is genetically predefined and modulated by many internal or external factors. One role of melanin is to protect the skin from harmful effects of sunlight and oxidative stress but unwanted pigmentation can produce a significant psychological stress [3]. Genetic and biochemical studies have identified several proteins that regulate melanin synthesis and the structural integrity of the melanosomal compartment. The best-characterized melanogenic proteins are tyrosinase, a copper-dependant glycoprotein, and its related proteins (tyrosine related protein-1 and 2; TRP-1 and TRP-2) [4]. Human tyrosinase was cloned and sequenced [5] and the deduced amino acid sequences revealed that tyrosinase is composed of 511 amino acids with more than 90% of the enzyme inside the melanosome. Tyrosinase catalyzes the hydroxylation of tyrosine to Dopa, which is the rate limiting step of melanogenesis and the oxidation of Dopa to Dopaquinone. TRP-1 has also been shown to play a crucial role in melanogenesis. The mature TRP-1 is the most abundant melanosomal protein [6] and TRP-1 shares with tyrosinase 70–80% nucleotide sequence homology and 40–45% amino acids identity. TRP-1 is transmembrane protein spanning melanosomal membranes [7], involved in regulating melanosomal maturation [8] and in contrast with mice, TRP-1 in human pigment cells do not display DHICA oxidase activity [9]. TRP-1 was shown to influence tyrosinase activity by forming a complex stabilizing tyrosinase [10, 11] and recent study indicated ethnic variation in TRP-1 expression suggesting its role in mediating differences in skin pigmentation in vivo [12]. In human cultured melanocytes, tyrosinase/TRP-1 complex formation has been demonstrated and this complex formation was mediated by Protein Kinase C beta (PKC beta) [13]. Molecular cloning revealed that PKC is a multigene family of at least 12 isoforms. Among them, PKC beta has specifically been reported to activate tyrosinase and human melanogenesis [14] by phosphorylating serine residues in its cytoplasmic domain [15] indicating that this entity regulates human melanogenesis at an early stage. Loss of PKC beta prevents melanogenesis in cultured pigment cells [15] and topical application of pan-PKC inhibitor has just been reported to reduce skin and hair pigmentation [16].Among the principal manifestations of photodamage in Asian women's skin, solar lentigo is a common benign pigmented spot on sun-exposed skin especially on the back of the hands, arms and on the forehead. For investigating this event, firstly, the evaluation on Japanese women's skin of the TRP-1 expression in solar lentigo spots in comparison with normal skin was performed and secondly, the study of the messenger RNA expression inhibition coding for tyrosinase/TRP-1 complex and/or PKC beta-I on melanogenesis was carried out. This antisense strategy is based the use of sequence specific oligodeoxyribonucleotides targeted to messenger RNA for modulating the expression of the enzymes instead of a direct inhibition after enzyme synthesis. Antisense strategy is a very specific approach, occurring at an upstream level of melanogenesis, with activity at nanomolar concentration without cytotoxicity. This strategy, targeting messenger RNA, offers the only way to specifically inhibit PKC beta-I, isoform expressed in melanocytes.〈section xml:id="abs1-2"〉〈title type="main"〉Methods〈section xml:id="abs1-3"〉〈title type="main"〉TRP-1 expression studyBiopsies (normal and solar lentigo area) were performed at Laboratoires Dermscan, (Lyon, France) in accordance with ethical committee procedure. The group consisted of 10 healthy Japanese women leaving in France with age ranging from 29 to 55 years. Biopsies were fixed in formaldehyde and embedded in wax. TRP-1 detection was performed with Mel-5 primary mouse monoclonal antibody (Signet, Dedham, U.S.A.) and a biotinylated antibody. Revelation was carried out with a standard phosphatase alkaline-staining and quantified using a computer assisted image analysis (LEICA Qwin, Rueil-Malmaison, France). Stained cells were counted over the complete length of the section and reported to the corresponding length of basal epidermis.〈section xml:id="abs1-4"〉〈title type="main"〉Inhibition of the tyrosinase/TRP-1 active complex formation on human melanocytesThe normal human melanocytes (NHM) were cultivated in a growth medium (K-SFM Invitrogen, Cergy, France) supplemented with 50 μg mL-1 BPE, 5 ng  mL-1 EGF, 10 ng  mL-1 bFGF (Invitrogen) and 0.25 μg  mL-1 PdBu (Sigma, St Quentin Fallavier, France).NHMs were seeded in 96-well microplates (Falcon, Franklin Lakes, NJ, U.S.A.) in a proportion of 10 000 cells per well, in 200 μl of growth medium without metabolic activator. Phosphorothioate antisens oligonucleotides (designed from GeneBank accession number X51420 and X06318) were tested at 250 nM, 500  nM or 1 μM, every day during 5 days. Control sequences and kojic acid (530 μM) were used as negative control and positive control respectively. After treatment, cells were rinsed with PBS then 50 μL of lysis buffer 0.5% Triton in PBS were added to the wells and the plate was shaken for 1 hour at 4°C. The reaction was initiated by adding 50 μL of substrate (10 mML-DOPA, Sigma) to each well. DOPAchrome was measured at 450  nM every 2 min, for 1 h and at 37 °C with regular shaking, using an optical density reader (microplate reader 340 ATTC-SLT-Labinstrument, Grödig/Salzburg, Austria). The reaction rates were calculated and expressed as 10-4 OD units/min. Results of the DOPA-oxidase activity were normalized with cell viability measured with XTT assay.〈section xml:id="abs1-5"〉〈title type="main"〉Results〈section xml:id="abs1-6"〉〈title type="main"〉TRP-1 expressionTRP1 expression was determined by calculating the number of positive cells per millimetres of basal epidermis. TRP1 expression was restricted to melanocytes which were distributed at regular intervals along the base of epidermis (〈link href="#f1-12"〉Fig. 1). No expression of TRP-1 was observed in melanosomes which had been transferred to neighbouring keratinocytes. We found that solar lentigo skin exhibited 89% TRP-1 expression more than normal skin (P = 0.05).〈figure xml:id="f1-12"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_12_12:ICS_254_f1-12"/〉Immunolocalization of TRP-1 in human skin. (a) Representative image of TRP-1 expression in solar lentigo. (b) Quantification of TRP-1 on solar lentigo and normal skin for the 10 Japanese women. Statistical differences were evaluated using the paired Student's test. The data is presented as the mean of 10 biopsies.〈section xml:id="abs1-7"〉〈title type="main"〉Inhibition of tyrosinase/TRP-1 active complex formation in human melanocytesThe antisense oligonucleotides were studied with regard to their action on melanogenesis inhibition. The assay was based on measurement of the reaction rate for dopa-oxidase activity. A drop in the reaction rate compared with the control assay shows a decrease in the enzyme activity and consequently a reduction melanin formation. Using antisense oligonucleotides designed for targeting TRP-1 expression, PKC beta-I synthesis or both targets in combination, the reaction rate of the dopa-oxidase activity was studied (Fig. 2). Firstly, we observed a significant decrease in activity (­17%) as a result of antisense targeting tyrosinase/TRP-1 complex. Secondly, using a sequence specifically targeting PKC beta-I mRNA, a significant reduction of dopa-oxidase activity (­13%) was obtained. T

Notes: Summary Background Chronic exposure to ultraviolet (UV) radiation induces changes in the skin structure which are mostly found in the superficial dermis and at the dermal–epidermal junction. Keratinocytes and fibroblasts contribute both to the synthesis and to the degradation of the molecules important for the integrity of this skin site. While several studies have reported on alterations of dermal components and of the functions of fibroblasts in vivo and in vitro after UV exposure, recent data suggested that keratinocytes could be the main skin cell type involved in the photoageing process. Objectives In this study, we analysed the expression of two keratinocyte molecules namely, β1 integrin (a proliferation marker) and involucrin (a differentiation marker) in sun-exposed and sun-protected facial skin of 16 healthy patients undergoing facial lifting. Methods Methods included histology, immunohistochemistry and quantitative reverse transcriptase–polymerase chain reaction analysis. Results Sun-exposed skin displayed the characteristic morphological and molecular features of dermal photoageing, compared with sun-protected skin, including dermal elastosis, diminished fibrillin and type VII collagen expression. Analysis of the epidermis in sun-exposed vs. sun-protected skin showed no histological differences, but dramatic changes in the expression of β1 integrin and involucrin. In sun-exposed skin, expression of β1 integrin protein by epidermal basal cells was reduced, paralleling a downregulation of β1 integrin mRNA, whereas involucrin protein expression was greatly enhanced in the superficial epidermal cell layers. Interestingly, the ratio between involucrin and β1 integrin protein expression was consistently increased in sun-exposed skin sites. Conclusions Collectively these results demonstrate that epidermal homeostasis is impaired by chronic UV exposure, and define β1 integrin expression as a molecular marker of the epidermal photoageing process.

Notes: Background  Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis. Mast cells and macrophages, which are found in higher numbers in photoaged skin, have been implicated in this process.Objectives  To analyse the phenotype of haematopoietic-derived infiltrating cells in photodamaged skin.Methods  Chronically sun-exposed (preauricular) and control sun-protected (postauricular) skin was recovered from eight healthy subjects undergoing cosmetic surgery (facial lifting).Results  Histological analysis showed that sun-exposed skin harboured more infiltrating mononuclear cells than sun-protected skin. Cellular infiltrates were found at the periphery of areas of elastolysis around hair follicles in sun-exposed sites, whereas they were found in the interfollicular dermis around blood vessels and around hair follicles in sun-protected samples. Immunohistochemical analysis revealed an increased number of mast cells, macrophages and CD4+ CD45RO+ T cells in sun-exposed dermis as well as a higher number of CD1a+ dendritic cells in sun-exposed epidermis, compared with the sun-protected samples. Thus photoageing displays histological features of chronic skin inflammation. However, no molecular sign of inflammation was observed and we even found a decreased expression of interleukin-1β mRNA in sun-exposed compared with sun-protected sites. Furthermore, the patients' skin looked normal and did not display any clinical inflammation.Conclusions  Collectively, these data show that chronic ultraviolet irradiation induces alterations of innate immune cells which are recruited in sun-exposed skin without being activated.